UNC5C is a netrin receptor in which a rare coding mutation, T835M, was recently identified that predisposes to late onset (LO) Alzheimer's disease (AD). Our preliminary suggest that UNC5C T835M causes neuron loss in the presence of A? pathology. We hypothesize that UNC5C T835M predisposes to LOAD by making neurons more vulnerable to cell death induced by pathogenic A? and Tau. We will 1) define cell death pathways in 5XFAD and P301S-Tau mouse models of A? and Tau pathology, respectively, crossed to knockin (KI) mice in which UNC5C T835M is expressed endogenously; 2) identify cell death pathways in human induced pluripotent stem cell (hiPSC) neurons generated from UNC5C T835M patient fibroblasts; 3) use mass spectrometry (MS)-based proteomics to determine the comprehensive UNC5C T835M cell death proteome. In Aim 1, we will determine the molecular mechanism of UNC5C T835M-mediated cell death induced by AD- associated stressors in mouse primary neurons and hiPSC neurons in vitro. We will analyze the cell death pathway induced by A?42 and Tau P301S in UNC5C T835M KI mouse primary neurons and hiPSC neurons derived from human UNC5C T835M patient fibroblasts. Activation of cell death molecules will be assessed using biochemical and cell biological methods. We will attempt to prevent A?- and Tau-induced cell death using pharmacologic inhibition and genetic knockdown of key molecules to validate the cell death pathway in vitro. In Aim 2, we will determine the molecular mechanism of UNC5C T835M-mediated cell death induced by A? and Tau pathologies in vivo. We will analyze cell death in the brains of UNC5C T835M KI mice crossed to 5XFAD and P301S-Tau transgenic mouse models of A? and Tau pathologies, respectively, which also exhibit neuron loss. We predict that UNC5C T835M will decrease the age of onset and increase the severity of cell death in each model. We will assess cell death markers and AD pathologies using biochemical, histological, and behavioral methods. We will attempt to prevent cell death in each model using pharmacologic inhibition and genetic knockdown of key molecules to validate the cell death pathway in vivo. In Aim 3, we will identify the UNC5C T835M cell death pathway proteome induced by pathogenic A? and Tau using unbiased proteomics. We will perform multiplexed quantitative tandem MS-based proteomic analysis of cell extracts from UNC5C T835M and repaired isogenic hiPSC neurons exposed to A?42 or Tau P301S-AAV to define the hiPSC neuron cell death proteome. We will also use tandem MS to identify the cell death proteome in brains of UNC5C T835M KI/5XFAD and KI/P301S-Tau transgenic mice relative to control brains from mice metabolically labeled with 15N. Hits will be prioritized according to potential druggability, validated by immunoblot and immunohistochemistry, and targeted for genetic knockdown in 5XFAD or P301S-Tau mice to potentially prevent neuron loss. These Aims are expected to provide proof of concept for therapeutic targeting of the UNC5C pathway for prevention of neuron loss in AD.